DESCRIPTION: The 3D structural organization of the genome plays a key role in nuclear functions such as gene expression and DNA replication. One of the next grand challenges of biology is to determine the detailed 3D genome architecture and elucidate its functional implications. In this proposal, we aim to develop a suite of novel technologies for comprehensive structural and dynamic analyses of genomes, in two aspects: (1) the spatial interactions and higher-order organization of genomic DNA and (2) the functional organization of protein complexes driving the 3D folding of genomes. The new computational and experimental methods proposed herein integrate multiple experimental inputs and generate physical higher-order models of the 3D nuclear genome organization. Analysis of these models will yield new insights into the principles and structure/functions relationships of the genome's 3D organization in space and time. We have the following aims: (1) Develop technologies for mapping the relative spatial positions of genomic DNA in the nucleus: our focus will be on the three major technical barriers faced by all current mapping technologies, namely inefficient and potentially biased data acquisition, lack of temporal resolution, and missing higher-order contact information. (2) Develop technologies for deciphering the Protein Code of 3D genome organizations: we will employ a proven pipeline for the isolation of native protein complexes, but extensively optimized for the purpose of reading out the chromatin interactome surrounding each particular chromatin interacting region. (3) Develop technologies for modeling and analysis of 3D genome structures: we will develop an integrated platform for population-based modeling of 3D genome structures, and develop a series of computational tools to perform structure-function mapping on the 3D genomes. (4) Develop sold validation techniques for guiding the above technology innovations: we will develop a set of simple-to-implement and easy-to-interpret techniques to validate our novel technologies, and this techniques can be generally adopted by the community for similar validation purposes.